To advance our long-term goal of understanding the regulation of immunoglobulin isotype switching in human B lymphocytes, we have been studying three enhancer regions lying downstream of the human immunoglobulin C-alpha genes to determine how these regions integrate signals from the B cell milieu to regulate Ig expression in conjunction with Ig gene promoters. These three enhancers correspond to DNase I hypersensitivity (HS) sites designated HS3, HS12 and HS4 and lie near the 3' end of the large duplication in the human IgH locus. We previously found that two T cell signals - IL4 and CD40L - inhibit the activity of the HS12 enhancer in transient transfections in the human B cell line CL01 (of germinal center phenotype). To understand the physiologic context of this inhibition we have screened other regulatory regions of immunoglobulin genes and find that whereas the 3' enhancer of the human kappa immunologloblin locus is also inhibited by these agents, the heavy chain intronic enhancer is minimally affected by these agents and the kappa intronic enhancer is actually stimulated by CD40L. V region promoters of the kappa and heavy chain loci are inhibited by both IL4 and CD40L. We have tried to localize potential binding sites for transcription factors that might mediate some of these regulatory effects using ligation-mediated polymerase chain reaction (LMPCR) technology. This method has revealed several positions from the human HS12 enhancer that show altered reactivity with dimethyl sulfate (DMS) in vivo in CL01 in comparison to naked DNA, and that are thus candidates for binding motifs of transcription factors. We will expand our panel of HS12 enhancer mutants to find out whether these regions of the HS12 sequence contribute to its activity. So far, the pretreatment of CL01 cells with IL4 and CD40 has been associated with only minor alterations in the LMPCR pattern detected with in vivo DMS treatment. We are also trying to understand the importance of a conserved palindromic structure that flanks the HS12 enhamcer. This palindromic structure flanking HS12 is conserved in rat and mouse, even though the actual sequence of the palindrome is not conserved. Reporter plasmids that contain or lack the entire palindrome or part of it have been constructed and are being analyzed for enhancer activity in transient and stable transfections. In collaborative projects with the labs of Dr. Richard Hodes and Dr. Andre Nussenzweig of the NCI we are studying the effects of several murine mutations on immunoglobulin gene recombination in vivo and in vitro. Mice carrying defective ATM genes were found to have defects in switching to IgG1 and IgE; switching defects were also seen in cultured B cells from these mice. Mouse cells with defective Ku80 genes displayed chromosome aberrations and poor growth in vitro. Mice with combined defects in Ku80 and p53 genes developed B cell tumors with chromosomal translocations involving the IgH locus and the c-myc gene.